Identification of essential residues for the biosynthesis of momilactone intermediate diterpene in wild rice Oryza officinalis

It is well-known that momilactones are diterpenoids that play important roles in the survival strategy of cultivated rice (Oryza sativa). Momilactones serve not only as phytoalexins against pathogens but also as allelopathic substances against other plant species. It has been reported that several wild rice species also produce momilactones. Among them, the CC genome species Oryza officinalis possesses two homologs of the diterpene synthase OsKSL4, which is responsible for momilactone biosynthesis: OoKSL4-1 and OoKSL4-2. Diterpene synthases have a role in constructing specific carbon skeletons in diterpenoid biosynthetic pathways. Previously, it was reported that OoKSL4-2, but not OoKSL4-1, converts syn-copalyl diphosphate to 9β-pimara-7,15-diene, the biosynthetic intermediate diterpene of momilactones. We herein identified residues that are required for the production of 9β-pimara-7,15-diene by comparing the sequences of OoKSL4-1 and OoKSL4-2. Amino acid substitution experiments reveal that the product profile of the triple substitution mutant OoKSL4-1-I592L/R700G/I702T was almost identical to that of OoKSL4-2, with converse mutagenesis further indicating that 585L, 693G, and 695T in OoKSL4-2 are important for the production of 9β-pimara-7,15-diene. Protein-ligand structure predictions suggest that the OoKSL4-1 triple mutant and OoKSL4-2 bind the substrate in a similar orientation within the binding pocket, positioning the two carbon atoms involved in the cyclization reaction close together. In contrast, wild-type OoKSL4-1 binds the substrate in a different orientation, resulting in a greater distance between the two carbons. This difference in the distance is well correlated with more efficient production of 9β-pimara-7,15-diene by OoKSL4-2 compared with OoKSL4-1.